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| United States Patent |
5,107,066
|
|
Ashikawa
, et al.
|
April 21, 1992
|
Method of producing potato cyst nematode hatching stimulus
Abstract
Disclosed is a novel method for the production of a substance which
accelerates the hatching of potato cyst nematode eggs using root cells of
plants of Solanaceae family.
In this method, the cultivation of the root cells may be carried out in a
specific medium, or, alternatively, the cells obtained by transformation
of the root cells with Ri plasmid T-DNA is used.
The disclosed method can be applied for the prevention of the damage by the
cyst nematodes in potato cultivation.
| Inventors:
|
Ashikawa; Ikuo (Tsukuba, JP);
Murai; Akio (Sapporo, JP);
Fukuzawa; Akio (Sapporo, JP);
Koshi; Masato (Tokyo, JP);
Kamada; Hiroshi (Tsukuba, JP)
|
| Assignee:
|
Harima Chemicals, Inc. (Kakogawa, JP)
|
| Appl. No.:
|
746096 |
| Filed:
|
August 12, 1991 |
Foreign Application Priority Data
| Dec 21, 1989[JP] | 1-329434 |
| Oct 02, 1990[JP] | 2-263209 |
| Current U.S. Class: |
119/6.7; 424/DIG.12; 435/41; 435/171; 435/252.2; 435/252.3; 435/253.6; 435/469; 800/294 |
| Intern'l Class: |
C12N 015/00; C12N 001/20; C12R 001/41 |
| Field of Search: |
424/93,DIG. 12
435/41,171,253.6,252.3,240.54,252.2,240.4
800/205
935/30,56,67,64
|
References Cited
U.S. Patent Documents
| 3581686 | Jun., 1971 | Raymond | 514/765.
|
| 4036987 | Jul., 1977 | Thompson et al. | 514/671.
|
| 4073939 | Feb., 1978 | Thompson et al. | 514/625.
|
| 4273768 | Jun., 1981 | Kochansky et al. | 514/141.
|
| 4658082 | Apr., 1987 | Simpson et al. | 435/320.
|
| 4801154 | Jan., 1989 | Stuart et al. | 435/240.
|
| 4863863 | Sep., 1989 | Cocking et al. | 435/172.
|
| 4886753 | Dec., 1989 | Marcker et al. | 435/172.
|
| 4990607 | Feb., 1991 | Katagiri et al. | 435/172.
|
| Foreign Patent Documents |
| 1272989 | Nov., 1989 | DE.
| |
| 0237784 | Oct., 1988 | JP.
| |
| 0245687 | Oct., 1988 | JP.
| |
| 0254982 | Oct., 1988 | JP.
| |
Other References
DPerwent Abs. 87-300111/43 Kamata et al. DE 3706978 (Oct. 1987).
Biotech Abs 87-01445 (J61221140) Oct. 1986.
Biotech Abs 86-02352 Ooms et al. PMBIDB "Plant Mol. Biol", (1985) 5,4,
205-212.
Patent Abstracts of Japan unex.applns. C field vol. 7, No. 143, Jun. 22,
1983; The Patent Office Japanese Government p. 58 C172 Kokai No. 58-55
493.
Patent Abstracts of Japan, unexamined applications C field, vol. 13, No. 49
Feb. 3, 1989, The Patent Office Japanese Government p. 153 C 565 Kokai-No.
63-245 687 (Tsumura & Co.).
Patent Abstracts of Japan, unexamined applications, C Filed, vol. 13, No.
63, Feb. 13, 1989, Kokai No. 63-254-982.
|
Primary Examiner: Lilling; Herbert J.
Attorney, Agent or Firm: Cushman, Darby & Cushman
Parent Case Text
This is a continuation of application No. 07/629,846, filed on Dec. 19, 199
0
Claims
We claim:
1. A method of stimulating hatching of potato cyst nematode eggs comprising
the steps of:
i) introducing into a Solanaceae family plant T-DNA in a Ri-plasmid of
Agrobacterium rhizogenes under conditions such that transformed root cells
of the Solanaceae family plant are obtained;
ii) cultivating said transformed root cells on a solid medium;
iii) inoculating said cells with a liquid culture medium;
iv) cultivating said cells;
v) collecting the cultivated cell medium; and
vi) contacting said medium with said eggs.
2. The method as set forth in claim 1, in which the plants of Solanaceae
family are selected from those of genera Solanum and Lycopersicon.
3. The method as set forth in claim 1, in which the liquid culture medium
contains 10-100 mM nitrate ions and no ammonium ion.
4. The method as set forth in claim 1, wherein said Agrobacterium are
removed from said transformed root cells prior to cultivating in step
(ii).
5. The method as set forth in claim 1, wherein said Agrobacterium
rhizogenes is Agrobacterium rhizogenes 15834 (ATCC 15834) or Agrobacterium
rhizogenes A4 (ATCC 43057).
Description
FIELD OF THE INVENTION
The present invention relates to a method of producing a substance for
stimulating hatching of potato cyst nematodes (cisto nematoda), which
comprises the biosynthesis in plants of Solanaceae family by means of an
organ culture of roots of plants of Solanaceae family.
DESCRIPTION OF THE PRIOR ART
The potato cyst nematode (Globodera rostochiensis) is one of harmful worms
in agriculture, which is parasitic on the plant of Solanaceae family such
as potato and tomato, and the damage is particularly serious in European
countries. In this nematode, a female imago turns into a cyst maintaining
eggs inside the body. The eggs are protected from various exterior adverse
conditions (low temperature and dryness) so as to exert extreme stability;
thus it is so far believed to be almost impossible to destroy the dormant
larvae within the eggs, which are protected by the cyst and shells, by the
use of agricultural chemicals.
One of the characteristics of the nematode is that the hatching
(termination of its dormant stage) is stimulated by a chemical substance
secreted from the roots of the parasitic plants. This fact has already
been found by Bannacke, but the responsible substance has not been
identified up to date.
This substance (or an extract containing this substance) can be applicable
as an agricultural chemical for against the potato cyst nematodes. Namely,
if this substance is distributed on a field in autumn after host plants
die off or in spring when the host plants are not yet planted in the
field, the dormant stage of the eggs of the cyst nematode is disturbed and
the hatching starts. Consequently, the resultant hatched larvae cannot
survive in the absence of the host to parasitize.
Recently, techniques for production of such valuable substances which is
produced by plants, using tissue cultures of plants, have been
considerably established. By culturing plant calluses, the valuable
substances can be obtained in a short period of time without waiting the
growth of corresponding natural plants. The production of shikonin by the
callus culture of gromwell (Lithospermum ervthrorhizon) has already been
industrialized.
However, in general, it is difficult to produce the substances, which are
specifically produced by plant organs, by using callus cultures. For the
production of such substances, cultures of plant organs are applicable.
For example, substances which are produced specifically in roots are
produced by root cultures. Several attempts have already been made to
obtain substances which are produced in plant roots using cultured roots
(Japanese Patent Published No. 245687/1988, Japanese Patent Published No.
237784/1988).
On the other hand, it is known that hairy roots are induced when
dicotyledonous plants are infected with Agrobacterium rhizogenes. These
hairy roots are transformed ones which grow extremely fast as compared to
corresponding untransformed roots and, in addition, are potentially able
to produce root-derived secondary metabolites and thus suitable for
industrial production of substances which are to be produced specifically
in roots. Examples of the substances produced using hairy roots includes
shikonin (Japanese patent Published No. 133995/1988) and ginsenosides
(Japanese Patent Published No. 254982/1988).
The Means to Solve the Problem
Cultured roots of plants of Solanaceae family can be obtained by the
following methods:
1. Seeds of plants are aseptically sown on a solid medium for plant culture
for germination, and then the roots of the seedlings are excised and
subcultured on the medium.
The medium used herein contains a carbon source, nitrogen sources, mineral
salts, metals in a trace amount, vitamins and others.
Examples of the carbon source include carbohydrates or fatty acid
derivatives. Examples of the preferable carbon source include glucose,
sucrose and analogs thereof Examples of the nitrogen source include
nitrate ions, ammonium ions and amino acids. Examples of the mineral salts
include phosphates, chlorides and sulfates Known examples of preferable
media include Murashige-Skoog medium, Gamborg medium, White medium,
Linsmaier-Skoog medium and Heller medium.
Phytohormones may be added to the above-mentioned media if necessary.
Furthermore, in order to produce a solid medium, for example, agar at a
concentration of 0.1%-2% by weight is added to an ordinary fluid medium.
In this case ammonium ion is not removed since the germination of seeds is
aimed at.
2. A part of a plant is cut off, sterilized and then placed on a medium
with or without phytohormones. Adventitious roots generated from the plant
are cut off and subcultured on the medium.
3. A part of a plant is cut off, sterilized and then planted on a medium
with phytohormones.
After several weeks, calluses are induced from the plant segment and
furthermore adventitious roots are induced from the calluses. These
adventitious roots are cut off and then subcultured on the medium.
Some of the cultured roots of the plants obtained according to the
above-mentioned method can be grown eternally by subculturing on a medium
with or without hormones.
In order to efficiently produce a substance to promote the hatching of
potato cyst nematodes, cultured roots of plants of Solanaceae family,
which have been subcultured on a solid medium, are inoculated in a liquid
medium for culturing. The medium to be used primarily contains, as to the
inorganic component of the medium except for inorganic nitrogen,
constituents of any known inorganic salt synthetic medium supplemented
with vitamins, amino acids and organic substances or the like. The present
invention is also characterized in that the medium to be used contains
10-100 mM nitrate ion and no ammonium ion as to the inorganic nitrogen
source. The amount of the substance for stimulating hatching of potato
cyst nematodes, which is produced in the cultured roots, decreases in a
medium containing ammonium ion.
When hairy roots are employed, plants of Solanaceae family are treated with
Agrobacterium rhizogenes and the induction is carried out by transforming
the plant cells by T-DNA of Ri-plasmid. In the present invention, any
Solanaceae plant can be used; however, the plants which belong to genus
Lycopersicon or Solanum are preferably used.
Examples of Agrobacterium rhizogenes used for forming hairy roots in these
plants include:
Agrobacterium rhizogenes 15834 (ATCC 15834)
Agrobacterium rhizogenes A4 (ATCC 43057)
According to the present invention, when plants are treated with
Agrobacterium rhizogenes, T-DNA in the Ri-plasmid of A. rhizogenes is
introduced (transformed) in the nucleus DNA of the plant cells.
In order to introduce the Ri-plasmid T-DNA in stalks, roots, leaves or
calluses of the above-mentioned plants of Solanaceae family to obtain the
transformed hairy roots, for example, the following methods can be used.
1. A method of direct inoculation to the plants
2. A leaf disk method using leaf segments (e.g., R. B. Horsh et al. Science
227, 1229, 1985)
The T-DNA is introduced by the above-mentioned method and then
Agrobacterium is removed from the hairy-roots. The bacteria were removed
by subculturing on media containing antibiotics.
The hairy roots thus obtained are cultured aseptically on an ordinary
medium for tissue culture. The medium used for the culture contains a
carbon source, nitrogen sources, mineral salts, metal in trace, vitamins
and so forth.
Examples of the carbon source are carbohydrates or fatty acid derivatives.
Examples of the preferable carbon source include glucose, sucrose and
analogues thereof. Examples of the nitrogen source include nitrate ion,
ammonium ion and amino acids. Examples of mineral salts include
phosphates, chlorides and sulfates. Known examples of preferable media
include Murashige-Skoog medium, Gamborg medium, White medium
Linsmaier-Skoog medium and Heller medium.
Phytohormones may be added to the above-mentioned media if necessary.
The amount of plants to be inoculated onto a medium varies in a wide range;
however, in general, about 10 mg to about 1 g fresh weight of either
cultured roots or hairy roots per 50 ml of a fluid medium is preferably
inoculated.
In the present invention, the cultivation is carried out at about
10.degree. C. to about 35.degree. C., preferably at 23.degree. C. to
28.degree. C. A substance for stimulating the hatching of potato cyst
nematodes is produced and accumulated in the medium or in the cultured
roots after the completion of the cultivation.
A test for the secretion of the substance for stimulating the hatching of
potato cyst nematodes into the fluid medium is carried out as follows:
Cysts of potato cyst nematodes were sieved from cyst-containing field
soil. The cysts are stored in water for about 10 days. The temperature is
preferably at 25.degree. C. The outer shells of the stored cysts are
broken using a bamboo spit so as to discharge the eggs. The eggs are
sifted out with a sieve and about 100 eggs each are placed on a Syracuse
lens dish. A fluid in which the above-mentioned root culture solution is
appropriately diluted and water are added to this lens dish to make the
final volume to 10 ml. The dilutions of the cultured medium are made
generally in the range of 1/10 to 1/100,000. An egg suspension with this
solution to be tested is allowed to stand in an incubator at 25.degree.
C., and after 10 days the hatching is observed using a microscopy to
calculate the hatching rate (%).
EXAMPLE 1
Seeds of the tomato plant (Lycopersicon esculentum) were sterilized with a
bactericide such as sodium hypochlorite and then sown on Murashige-Skoog
(MS) solid medium containing 3% sucrose. The root tip segments of the
germinated sterile plant were excised and then subcultured on MS solid
medium to prepare tomato cultured roots.
Fifty milliliters each of a 1/2 MS liquid medium (A) in which
concentrations of major salts in MS were reduced to 1/2 and a modified MS
liquid medium (modified 1/2 MS, B) in which all the nitrogen components of
the major salts, except ammonium ion, were replaced by KNO.sub.3 was
placed in a 200-ml volume of Erlenmeyer flask and then sterilized at
120.degree. C. for 15 minutes. On each of these liquid media, about 100 mg
of the above-mentioned tomato cultured roots was inoculated and then
cultured at 25.degree. C. for 20 days with shaking (rotating at 70
rpm/minute).
When the cultured roots were cultured in the 1/2 MS medium (A), the
hatching activity was observed up to 1000-fold dilutions; when cultured in
the modified 1/2 MS medium (B), the production of the hatching
accelerating substance increased about ten times so that the hatching
activity was observed even when the medium was diluted 10,000 times.
On the other hand, in order to detect the hatching substance remaining in
the cultured roots after the cultivation, the cultured roots cultivated in
the modified 1/2 MS medium were homogenized in 10 ml of water and then the
supernatant was taken for the determination of the hatching activity The
hatching activity was observed down to the 1/1,000 dilution (Table 1).
This value is considerably low as compared to that observed in the medium
after cultivation, which indicates that the hatching stimulus is mostly
released from the roots to the medium.
TABLE 1
______________________________________
Hatching rates (%) in tomato cultured root media
and in cultured root extract
1/100 1/1000 1/10000 1/100000
______________________________________
1/2 MS liquid medium (A)
84.9 55.5 20.0 25.0
Modified 1/2 MS liquid
79.1 78.7 50.7 22.8
medium (B)
Cultured root extract
71.2 60.5 27.0 16.9
______________________________________
EXAMPLE 2
(1) Tomato hairy roots
Seeds of the tomato plant (Lycopersicon esculentum) were sterilized with a
bacericide such as sodium hypochlorite and then seeded on a
Murashige-Skoog (MS) solid medium containing 3% sucrose. Stems of the
sterile seedlings were inoculated with Agrobacterium rhizogenes A4 (ATCC
43057) carrying Ri-plasmid.
After 2 to 4 weeks, the hairy roots generated from the inoculation site
were excised, transferred on an MS solid medium containing 0.5 g/l of
claforan and then transferred to a fresh medium of the same composition
after 1 to 2 weeks. This procedure was repeated 2 to 3 times and then the
sterile hairy roots were obtained.
Fifty milliliters each of a 1/2 MS liquid medium in which concentrations of
the major salts in MS were reduced to 1/2 and an MS liquid medium was
placed in a 200-ml volume of Erlenmeyer flask and then sterilized at
120.degree. C. for 15 minutes. On each of these fluid media 20 mg of the
above-mentioned hairy roots was inoculated and then cultured at 25.degree.
C. for 20 days with shaking (rotating at 70 rpm/minute).
The medium after the cultivation was diluted with water and then added to
the suspension of the eggs of potato cyst nematodes. The hatching activity
in the suspension was observed.
The results (hatching rates) are shown in Table 2.
It is shown that the hatching activity was observed even when the medium
was diluted 10,000 to 100,000 folds.
On the other hand, in order to detect the hatching stimulating substance
remaining in the hairy roots after the cultivation, the hairy roots were
homogenized in 10 ml of water and then the supernatant was taken for the
determination of hatching activity. The hatching activity was observed in
down to the 1/1,000 dilution (Table 1). However, the activity was
extremely low as compared to that in the culture fluid after the
cultivation, which indicates that the hatching stimulating substance is
mostly released from the roots to the medium.
TABLE 2
______________________________________
Hatching rates (%) of tomato hairy root media
and cultured root extract
Dilution of fluid for detection
1/100 1/1000 1/10000 1/100000
______________________________________
MS hairy root medium
71.4 83.5 81.9 26.0
1/2 MS hairy root medium
71.2 79.8 58.9 70.6
MS hairy root extract
81.7 59.6 29.5 12.9
______________________________________
(2) Potato hairy roots
A stalk from the potato plant (Solanum tuberosum) in its early stage grown
from the tuber of the plant was cut off, sterilized and infected with
Agrobacterium rhizogenes A4 on the section of the stalk. When this stalk
was grown aseptically on an MS medium, hairy roots were generated from the
inoculation site 2 to 4 weeks after the infection. The hairy roots were
excised and then subcultured on a solid MS medium for sterilization as
described above for the tomato hairy roots.
The hairy roots were cultured in 50 ml of an MS fluid medium in a 200-ml
volume of Erlenmeyer flask as described above for the tomato hairy roots.
Table 3 shows the results of the test for their ability to hatch potato
cyst nematodes.
Differing from the tomato hairy roots, the hatching stimulating substance
is retained mostly in the roots rather than released in the medium.
TABLE 3
______________________________________
Hatching rates (%) of potato hairy root medium
and cultured root extract
Dilution of fluid for detection
1/100 1/1000 1/10000 1/100000
______________________________________
MS hairy root medium
73.9 56.1 28.5 16.7
MS hairy root extract
79.1 76.0 32.7 14.5
______________________________________
EFFECTS OF THE INVENTION
According to the present invention, a substance for stimulating the
hatching of potato cyst nematodes is effectively produced from the plants
of genus Solanaceae and genus Lycopersicon. The method of the present
invention is extremely effective to exterminate this nematodes.
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